Progressive fibrosis is a hallmark of interstitial lung diseases such as idiopathic pulmonary fibrosis. Fibrosis stiffens the lung parenchyma. Preliminary data provided in this proposal demonstrate that normal lung parenchymal tissue is more compliant than previously predicted, and that highly localized increases in the stiffness of fibrotic lesions are much greater than previously recognized. Fibroblasts grown on substrates as stiff as fibrotic lesions engage in rapid proliferation and abundant matrix synthesis; in marked contrast these behaviors are largely suppressed on substrates as compliant as normal lung tissue. The central hypothesis that we pose based on these data is that the mechanical environment present in lung fibrosis triggers a fibrogenesis program in resident fibroblasts that promotes feedback amplification of the disease. We propose four specific aims: (1) quantify the stiffness of the parenchyma in normal lung tissue and developing and established fibrotic lesions; (2) test whether the transition in matrix stiffness from normal to fibrotic levels is a necessary precondition for lung fibroblast proliferation and fibrogenic activation; (3) test the role played by cytoskeletal dynamics and serum response factor activation in driving stiffness-dependent fibroblast biology; and (4) identify key transcription factors coordinating fibroblast transitions between quiescent and fibrogenic states when transferred between compliant and stiff matrices. These aims will be carried out in novel 2D and 3D models that allow clear delineation of the effects of lung matrix stiffness on key fibrogenic behaviors of lung fibroblasts. Throughout the experimental plan we will examine the interplay between matrix stiffness and the soluble environment present in fibrosis as it impacts on fibroblast biology. This research will generate novel insights into the molecular mechanisms of stiffness-dependent fibroblast activation in fibrotic lungs, and identify critical regulators of fibrogenesis suitable for therapeutic targeting. PUBLIC HEALTH RELEVANCE: Lung fibrosis stiffens affected tissue. The experiments proposed here will test whether pathophysiological changes in stiffness promote fibrosis by stimulating lung fibroblasts. Understanding the role mechanical factors play in fibroblast activation could lead to new strategies to treat fibrosis.